Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM

Varshney, Dhaval; Petit, Alain-Pierre; Bueren-Calabuig, Juan A.; Jansen, Chimed; Fletcher, Dan A.; Peggie, Mark; Weidlich, Simone; Scullion, Paul; Pisliakov, Andrei V.; Cowling, Victoria H.

doi:10.1093/nar/gkw637

Cited by 62 publications

(80 citation statements)

References 34 publications

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“…Indeed, in the vaccinia virus family, the D1 catalytic subunit alone is unstable and inactive, and its stability and MTase activity are enhanced by the presence of D12 through the increase in GTP, SAM, and GpppA binding affinity (41,42). Similarly, the human RNA N7 MTase (RNMT) is allosterically activated by RNMT activating miniprotein that stabilizes its structure and favors the recruitment of methyl donors (43).…”

Section: Discussionmentioning

confidence: 99%

Binding of the Methyl Donor S -Adenosyl- l -Methionine to Middle East Respiratory Syndrome Coronavirus 2′- O -Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

Aouadi

Blanjoie

Vasseur

et al. 2017

J Virol

100

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The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S-adenosyl-L-methionine (SAM)-dependent 2=-Omethyltransferase (2=-O-MTase) that is thought to methylate the ribose 2=-OH of the first transcribed nucleotide (N 1 ) of viral RNA cap structures. This 2=-O-MTase activity is regulated by nsp10. The 2=-O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2=-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7m GpppG (cap-0) into 7m GpppG 2=Om (cap-1) RNA by 2=-OH methylation of N 1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2=-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2=-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2=-O methylation and to restore the host antiviral response.IMPORTANCE MERS-CoV codes for a cap 2=-O-methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2=O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2=-Omethyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/ nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2=-O-methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.KEYWORDS RNA processing, RNA virus, biochemistry

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Section: Discussionmentioning

confidence: 99%

Binding of the Methyl Donor S -Adenosyl- l -Methionine to Middle East Respiratory Syndrome Coronavirus 2′- O -Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

Aouadi

Blanjoie

Vasseur

et al. 2017

J Virol

100

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“…9,10 RNMT has a catalytic core, which is homologous in structure and function to other eukaryotic cap methyltransferases. 11 In addition, RNMT contains an N-terminal noncatalytic domain, which is conserved in mammals but not in other eukaryotes (Fig. 2).…”

Section: Regulation Of Mrna Cap Methylationmentioning

confidence: 99%

“…12 RAM activates RNMT by altering the position of key active site residues, increasing methyl donor binding. 11 RAM also has a high-affinity RNA binding domain which may increase the rate of recruitment of transcripts in general or may have specificity for particular transcript motifs. 10,13 In human cells, RNMT-RAM is recruited via the N-terminal domain of RNMT to RNA pol II proximal to transcription initiation sites.…”

Section: Regulation Of Mrna Cap Methylationmentioning

confidence: 99%

Regulation of mRNA capping in the cell cycle

Aregger

Cowling

2016

RNA Biology

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The mRNA cap structure, which is added to nascent RNA pol II transcripts, recruits the protein complexes required for pre-mRNA transcript processing, mRNA export and translation initiation. The enzymes which catalyze mRNA cap synthesis are regulated by cellular signaling pathways which impact on their expression, localization and activity. Here we discuss the recent observation that the mRNA cap methyltransferase, RNMT, is phosphorylated on Thr-77 by CDK1-cyclin B1, which regulates its activity and the proteins with which it interacts. RNMT Thr-77 phosphorylation provides a burst of mRNA cap methyltransferase activity during early G1 phase at a time when transcription is reactivated following completion of the cell cycle. This co-ordination of transcription and mRNA capping makes an important contribution to gene expression in the cell; preventing RNMT Thr-77 phosphorylation inhibits cell proliferation. Here we discuss these findings and how mRNA cap synthesis may be regulated in other scenarios.

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“…Additionally, it is also possible that as our truncated hRNMT is less selective as it lacks the hRNMT activating Miniprotein (RAM) binding domain and the RAM protein. Indeed it was previously demonstrated that RAM stabilizes the structure and positioning of the hRNMT lobe and increases the specific recruitment of SAM (Varshney et al, 2016). This high inhibition of hRNMT MTase activity may be overcome in the presence of its allosteric activator RAM, which may allow to stringent the specificity of the compounds.…”

Section: Discussionmentioning

confidence: 99%

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

et al. 2017

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Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases.

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Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM

Cited by 62 publications

References 34 publications

Binding of the Methyl Donor S -Adenosyl- l -Methionine to Middle East Respiratory Syndrome Coronavirus 2′- O -Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

Binding of the Methyl Donor S -Adenosyl- l -Methionine to Middle East Respiratory Syndrome Coronavirus 2′- O -Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

Regulation of mRNA capping in the cell cycle

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

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